1. Field of the Invention
The present invention relates to a process of preparing nucleic acid so as to recover nucleic acid as a sample for various procedures using nucleic acid, more-specifically to a process of preparing nucleic acid so as to recover the nucleic acids of bacteria and the like such as those of the genus Mycobacterium having been said to involve much difficulty in the extraction from biological samples such as feces having been said to involve much difficulty in the purification.
2. Description of the Related Art
(Extraction Process)
General processes of extracting nucleic acid from biological materials such as viruses, bacteria, fungi, protozoa, plants and animals have been used, such as a process of decomposing cell wall with enzymes such as lysozyme and extracting nucleic acid in solution and a process of decomposing protein with enzymes such as proteinase to destruct cell and then extracting nucleic acid in solution.
However, these extraction processes with enzyme treatment require full skills and times, so these processes are at poor treatment efficiencies, disadvantageously. When these extraction processes are applied to biological materials with hard cell wall, such as bacteria of the genus Mycobacterium and fungal spores, the cells cannot be decomposed. Therefore, the nucleic acid extraction efficiency of these processes is extremely low. So as to extract nucleic acid from bacteria of the genus Mycobacterium, which involve much difficulty in the destruction of the bacterial cells, the method described in the report of Vary, et al. (J. Clin. Microbiol., 28(5), 933-937, (1990)) requires treating the bacteria with enzymes of three species over 48 hours in total.
So as to overcome the problem that such bacterial cells have such hard cell walls that the bacterial cells involve difficulty in the destruction thereof, accordingly, a process has been known, which includes vigorous agitation of bacterial cells, using phenol and fine particles. For example, the report of Giessen, et al. (J. Med. Microbiol., 36(1992), 255-263) describes a modified approach including a step of destructing the bacterial cells through vigorous agitation together with fine particles of zirconium oxide in TE buffer (10 mM Tris-HCl, 1 mM EDTA) and phenol. Additionally, U.S. Pat. No. 4,918,178 includes a similar description.
However, the extraction process has an advantage of higher extraction efficiency as well as disadvantages of the use of a very hazardous reagent phenol and the pollution of laboratory environment with phenol during the agitation procedure. Therefore, the extraction process is poor in terms of practical applicability.
(Purification Process)
So as to purify nucleic acid from an extract solution of nucleic acid, proteins and lipophilic contaminants in the extract solution of nucleic acid should be removed from the extract solution. Generally, a process has been known, which includes the denaturation and removal of such protein and lipophilic contaminants using phenol/chloroform and the concentration and purification of the resulting nucleic acid fraction via ethanol precipitation and the like. Further, a process of adsorbing nucleic acid on glass beads and the like to remove ingredients except for nucleic acid and a process of filtering off contaminants using filter and the like have been known.
However, especially when a biological sample is feces and the like, the aforementioned purification process occurs problems as follows. For example, by the process of using phenol and chloroform, contaminants other than the lipophilic contaminants cannot be removed; by the process of using glass beads, contaminants in the feces inhibit the adsorption of the nucleic acid on the glass beads; and by the process of using filter, filtration cannot be accomplished due to clogging of the filter with contaminants in the feces. Therefore, nucleic acids with high purity cannot be obtained from feces by these methods.
Indeed, after a process for purifying nucleic acids was performed by treating feces as a sample with enzymes using proteinases followed by conducting the phenol-chloroform extraction, precipitates of the nucleic acids were obtained by an ethanol precipitation method. As a result, the precipitates were yellowish or brownish. This result indicates that contaminants in the feces are not removed and purification of the nucleic acids is not accomplished in high purity.
So as to recover highly pure nucleic acid from feces involving much difficulty in the purification of the nucleic acid therein, the inventors developed a process of purifying nucleic acid using quaternary ammonium salt (JP-A-2002-37798A). According to the process, contaminants derived from feces excluding nucleic acid can be removed at high efficiency.
So as to prepare nucleic acid from an intended sample, a process and a purification process appropriate for the properties of the sample are selected from these processes. Generally, the two processes for extraction and purification are carried out sequentially.
As described above, however, not any simple or efficient extraction process has been developed yet. Although the excellent nucleic acid purification process of the inventors has been proposed, the process should be done at two steps of extraction and purification so as to prepare nucleic acid. So as to rapidly carry out the treatment of a great number of samples, a nucleic acid preparation process for simultaneous purification together with simple and high efficient extraction has been desired.